Interleukin-1 deficiency in combination with macrophage depletion increases susceptibility to Pseudomonas aeruginosa bacteremia

We evaluated the role of IL-1 during Pseudomonas aeruginosa bacteremia by intravenously injecting P. aeruginosa strain D4 into IL-1-deficient and WT mice. The two strains showed equivalent mortality rates. However, when the mice were pretreated with cyclophosphamide, bacteremia-induced mortality was significantly greater in the IL-1-deficient mice than in the WT mice ( P < 0.01). We then investigated the role of neutrophils and macrophages in protecting IL-1-deficient mice from bacteremia by administering anti-Gr-1 antibody or liposomes containing dichloromethylene diphosphonate, respectively. After P. aeruginosa inoculation survival was significantly lower in the macrophage-depleted IL-1-deficient mice than in the WT mice. In contrast, neutrophil depletion did not have this effect. Compared to the macrophage-depleted WT mice, the macrophage-depleted IL-1-deficient bacteremic mice had higher bacterial counts in various organs 48 and 72 hr post-infection. They also had lower TNF-α, IL-6, and INF-γ concentrations in their livers during the early phase of sepsis. Thus, IL-1 deficiency becomes disadvantageous during P. aeruginosa bacteremia when it is accompanied by immunosuppression, particularly when macrophage functions are seriously impaired.     

Antimicrobial and antifouling efficacy of urinary catheters impregnated with a combination of macrolide and fluoroquinolone antibiotics against Pseudomonas aeruginosa

The incidence of catheter associated urinary tract infections (CAUTIs) is increasing worldwide. This study was designed to modify a biomaterial by impregnating a silicone urinary catheter with combination of a macrolide, azithromycin (AZM) and a fluoroquinolone, ciprofloxacin (CIP). Drug release profiles showed slow yet continuous release of antibiotics from catheters for one month. In vitro efficacy testing showed that group B catheters [3% (w v^?1) CIP + 6% (w v^?1) AZM] outperformed group A catheters [2% (w v^?1) CIP + 5% (w v^?1) AZM] by (1) showing larger zones of inhibition (>31 mm) compared to group A (<28 mm) for up to 30 days against Pseudomonas aeruginosa PAO1; (2) killing adhered bacteria in 24 h compared to 24–48 h in group A; (3) showing longer antimicrobial durability for four weeks; and (4) exhibiting a stable real-time shelf life of one year, suggesting that these catheters can be explored in clinical settings, especially in long-term CAUTI.     

大蒜素对铜绿假单胞菌生物膜早期黏附及胞外多糖的影响

目的研究大蒜素对铜绿假单胞菌PA01菌株生物膜（biofilm,BF）早期黏附及胞外多糖复合物（Extracellular Polymeric Substances,EPS）的影响。方法利用荧光多功能酶标仪检测各组不同时间点96孔板中pGF-Puv转化PA01菌株的荧光强度,计算黏附率以表示干预对不同时间点细菌黏附的影响,利用荧光显微镜下定性观察细菌的黏附量;应用异硫氰酸标记的刀豆蛋白A（FITC conjugated concanavalin A,FITC-conA）特异性结合细菌EPS,荧光显微镜下定性观察各组EPS的变化;利用硫酸-苯酚法定量各组细菌EPS的产量。结果6h组,大蒜素高浓度干预后细菌的黏附率由0．70±0．03下降至0．50±0．01,t=15．014,P〈0．05,大蒜素低浓度干预后黏附率也有下降,但不及高浓度组明显;除了9h组其他时间组趋势与6h组大致相似,可能与大蒜素含量下降有关。荧光显微镜观察可见生理盐水对照组细菌菌落分布,干预组黏附的细菌稀疏散在分布,以高浓度为甚。FITC-conA可使胞外多糖显色,在荧光显微镜下观察可见大蒜素干预后EPS减少,稀薄;EPS定量实验,EPS总量大蒜素高浓度干预组（181．19±1．59）μg较生理盐水对照组（602．66±21．94）μg有明显降低,t=60．589,P〈0．05。结论大蒜素可显著减少PA01菌株黏附及产EPS的能力。     

铜绿假单胞菌分泌性蛋白的抗肿瘤效应

目的 探讨3株不同来源的铜绿假单胞菌分泌性蛋白的抗肿瘤活性。方法 将3株不同来源的铜绿假单胞菌经LB培养基对其过夜静置培养,离心获得上清液,采用硫酸铵盐析沉淀蛋白质,再经PBS透析除盐。然后将蛋白作用于人肝癌细胞Hep-G2、宫颈癌细胞HeLa、肺癌细胞A549和人永生化表皮细胞HaCaT,CCK-8检测其对细胞的毒性作用,吉姆萨染色观察凋亡细胞形态变化。结果 SDS-PAGE证实成功获得不同分子量的分泌蛋白,CCK-8结果显示混合蛋白对3种不同肿瘤细胞的生长均有不同程度的抑制作用,且其抑制作用存在一定的时间和浓度依赖性,但对人正常细胞无明显抑制作用。吉姆萨染色初步显示细胞形态为凋亡状态。结论 初步证实该3株铜绿假单胞菌产生的分泌性蛋白具有不同程度的抗肿瘤作用,为进一步分离纯化具有抗肿瘤活性的单一蛋白质及研究其抗肿瘤机制提供依据。     

Synergistic activity of chlorhexidine and synoeca-MP peptide against Pseudomonas aeruginosa

‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬This study aims to evaluate the in vitro antimicrobial and immunomodulatory activities and cytotoxicity of chlorhexidine (CHX) and synoeca-MP peptide alone or in combination against Pseudomonas aeruginosa. The antimicrobial property was evaluated by the determination of minimal inhibitory concentration, minimum bactericidal concentration, and planktonic bacteria and biofilm inhibition. Immunomodulatory activity was determined by enzyme-linked immunosorbent assay and nitric oxide production by the Griess reaction method. According to the results, synoeca-MP combined with CHX demonstrated antimicrobial effectiveness compared with its isolated use, in addition to immunomodulatory activity (upregulating MPC-1 and tumor necrosis factor-α and downregulating nitric oxide and interleukin-10). In this context, it is expected that the substances, together, could be capable of controlling bacterial infection and dissemination, besides potentiating macrophages’ immune response against the studied microorganism. Moreover, reducing the CHX concentration by the addition of synoeca-MP peptide may, in a beneficial way, minimize the undesirable effects of both, CHX and synoeca-MP in a clinical setting.     

铜绿假单胞菌医院感染调查及耐药分析

目的探讨铜绿假单胞菌(PA)医院感染的临床分布及耐药性,为临床合理使用抗菌药物及控制医院感染提供依据。方法 PA的培养与鉴定严格按照《全国临床检验操作规程》进行。药敏试验采用纸片扩散法(K-B法)。结果收集的359株PA中,分离率居前3位的临床科室依次为ICU、呼吸内科和神经内科,分别占38.1%、17.5%和13.9%;临床标本中以呼吸道标本分离率最高,占62.7%;PA对头孢哌酮/舒巴坦的耐药率最低(9.7%),其次为阿米卡星(13.1%)和头孢他啶(17.8%),另12种抗菌药物的耐药率均较高。结论 PA耐药性已十分严重,临床应根据药敏结果合理使用抗菌药物并加强对细菌耐药性的全面监测。     

QS系统中LasR和RhlR蛋白对铜绿假单胞菌生物被膜形成的影响以及免疫原性研究

为探讨铜绿假单胞菌 PAO1 中 lasR 和 rhlR 基因表达产物的分子生物学特性,研究它们对铜绿假单胞菌生物被膜形成的影响以及对小鼠的免疫保护效果,采用聚合酶链式反应 (PCR) 方法扩增铜绿假单胞菌标准株 PAO1 中的 lasR 和 rhlR 基因,全自动荧光测序仪测序,并用 Blast 方法检测克隆片段. 利用 pGEX4T-1 载体分别构建 lasR/rhlR-pGEX4T-1 重组质粒,在大肠杆菌 BL21(DE3)中诱导表达,并经过免疫印迹实验验证其生物学活性. 用硅胶膜培养法建立生物被膜模型,诱导转入了pGFPuv 质粒的铜绿假单胞菌 PAG0305 形成生物被膜,并测定 LasR 蛋白和 RhlR 蛋白对生物被膜形成的影响. 同时用纯化的重组蛋白免疫小鼠,菌落计数法检测免疫组和对照组鼠肺对铜绿假单胞菌的清除率. 以 PAO1 染色体 DNA 为模板的 PCR 结果显示,lasR 的全基因序列为 720 bp,rhlR 基因序列为 726 bp,经序列分析和同源性比较分别与 GenBank 中 lasR/rhlR 基因(登录号:M59425; AE004768) 的同源性为 100%. 大肠杆菌 BL21 (DE3) 分别转化重组质粒 lasR/rhlR-pGEX4T-1 后,经 IPTG诱导和 SDS- 聚丙烯酰胺凝胶电泳分析,表达的融合蛋白分子质量均为 54 ku 左右,与预期蛋白质分子质量相同. 荧光显微镜观察和测定结果表明,在硅胶膜上 PAG0305 能够形成典型的发荧光的生物被膜,LasR 或 RhlR 蛋白 (10 mg/L) 存在的情况下,PAG0305 生物被膜的形成速度在前三天比对照组平均提高 40.77%,而且两蛋白单独存在与同时存在时的作用相同. 体内实验中,免疫小鼠肺部对铜绿假单胞菌的清除率显著高于未经免疫的正常组 (P < 0.05). 上述结果表明:构建的lasR/rhlR-pGEX4T-1 重组质粒能够在大肠杆菌 BL21(DE3)中成功地表达并具有生物学活性. LasR/RhlR 蛋白在体外模型中能够加快铜绿假单胞菌生物被膜的形成速度,是调节铜绿假单胞菌生物被膜形成的重要因素之一. 免疫结果表明,重组蛋白对小鼠表现出一定的保护作用,这为进一步开展疫苗研究奠定了基础.     

Reduction of antibiotic-induced biofilm accumulation of Pseudomonas aeruginosa by quaternized phytoglycogen

Biofilms are oft cited as a factor in the unwanted persistence and recalcitrance of microbial life and a strong research initiative exists to identify, understand, and target vulnerabilities. Phytoglycogen is a biodegradable nanoparticulate biomaterial that is purified from crop plants. Importantly, the highly branched glucan structure provides a scaffold on which to construct novel polymers. Functionalized phytoglycogen (FP) was synthesized using green chemistry principles. Screening of several molecules identified a form of quaternized phytoglycogen which reduced biofilm formation and accretion by Pseudomonas aeruginosa. Exposing P. aeruginosa to modified phytoglycogen and antibiotic in combination not only substantively reduced biofilms, but also prevented increased biofilm formation, a biological response to suboptimal antibiotic concentrations. Treatment of pregrown biofilms with sub-minimum inhibitory concentration antibiotic alone also led to increased proliferation, whereas FP-antibiotic combinations prevented or reduced the extent of this. Swimming, swarming and twitching motility, all critical for biofilm development, were negatively affected by FP. This work supports phytoglycogen as a promising foundational molecule for novel polymers, including those with anti-biofilm function. Critically, in addition to published reports on how suboptimal antibiotic concentrations promote biofilm formation, we demonstrated a similar effect upon pre-existing biofilms, indicating a further route for the failure of antibiotic therapies.     

Contribution of multidrug efflux pumps to multiple antibiotic resistance in veterinary clinical isolates of Pseudomonas aeruginosa

The contribution of efflux pumps to multidrug resistance in 12 Pseudomonas aeruginosa isolates from various animal sources was assessed. Western immunoblot analyses demonstrated that all twelve isolates expressed significant levels of the MexAB OprM efflux system whereas two isolates simultaneously expressed the MexEF OprN or MexXY systems, respectively. One strain contained a single mutation in mexR, a regulator of mexAB-oprM expression, that did not adversely affect the MexR amino acid sequence, and three isolates contained the same, single base change in the mexA-mexR intergenic region. The MexXY-expressing strain contained two base substitutions in its mexZ regulatory gene which did not alter the MexR sequence.     

Saccharides of seven Pseudomonas aeruginosa immunotypes

Abstract The structures of O-specific polysaccharides obtained by mild acid degredation of lipopolysaccharides (LPS) from seven Pseudomonas aeruginosa Fisher's immunotypes have been studied. The polysaccharides consist mainly of monoamino and diamino sugars, frequently also carrying acidic functions. Some of the sugars were detected in nature for the O-specific polysaccharides of the immunotypes 2, 3, 4, 5 and 6 are identical to those of the polysaccharides of the 011; 0(2a)2c; 01; 010a, 10b and 07a, 7d Lányi-Bergan serological subgroups respectively, whereas no analogues have been found for the immunotypes 1 and 7. Some cross-reactions between the LPS of different immunotypes were observed in passive haemagglutination tests; the results of inhibition of passive haemagglutination and agar gel immunoprecipitation point, however, to a specificity of the LPS. Many of the LPS of the seven Pseudomonas aeruginosa immunotypes manifest rather a high cross-protective activity in active immunization tests in mice. The nature of the cross-protective activity of the LPS is discussed.     

铜绿假单胞菌及L型诱导巨噬细胞凋亡的实验研究

目的研究铜绿假单胞菌（PA）及L型诱导巨噬细胞凋亡的能力,比较二者的差异。方法用生物素断端标记（TUNEL）法检测PA及L型感染巨噬细胞2、4、8、12、16和20h后各时间段的细胞凋亡率,Giemsa染色观察细胞凋亡情况,硝酸还原酶法检测培养液中一氧化氮（NO）的浓度变化。结果 PA及L型能诱导巨噬细胞发生凋亡,与对照组比较差异有统计学意义（P〈0.05）;L型诱导细胞的凋亡率弱于原菌（P〈0.05）;PA及L型感染组培养液NO浓度较对照组明显升高（P〈0.05）。结论 PA及L型可诱导巨噬细胞发生凋亡,L型较其原型诱导细胞凋亡的能力弱,NO可能在巨噬细胞凋亡中发挥一定作用。PA及L型可通过诱导巨噬细胞凋亡,发挥致病作用。     

Magnetic fields suppress Pseudomonas aeruginosa biofilms and enhance ciprofloxacin activity

Due to the refractory nature of pathogenic microbial biofilms, innovative biofilm eradication strategies are constantly being sought. Thus, this study addresses a novel approach to eradicate Pseudomonas aeruginosa biofilms. Magnetic nanoparticles (MNP), ciprofloxacin (Cipro), and magnetic fields were systematically evaluated in vitro for their relative anti-biofilm contributions. Twenty-four-hour biofilms exposed to aerosolized MNPs, Cipro, or a combination of both, were assessed in the presence or absence of magnetic fields (Static one-sided, Static switched, Oscillating, Static + oscillating) using changes in bacterial metabolism, biofilm biomass, and biofilm imaging. The biofilms exposed to magnetic fields alone exhibited significant metabolic and biomass reductions (p < 0.05). When biofilms were treated with a MNP/Cipro combination, the most significant metabolic and biomass reductions were observed when exposed to static switched magnetic fields (p < 0.05). The exposure of P. aeruginosa biofilms to a static switched magnetic field alone, or co-administration with MNP/Cipro/MNP + Cipro appears to be a promising approach to eradicate biofilms of this bacterium.     

The influence of flow cell geometry related shear stresses on the distribution,structure and susceptibility of Pseudomonas aeruginosa 01 biofilms

The effects of non-uniform hydrodynamic conditions resulting from flow cell geometry (square and rectangular cross-section) on Pseudomonas aeruginosa 01 (PAO1) biofilm formation, location, and structure were investigated for nominally similar flow conditions using a combination of confocal scanning laser microscope (CSLM) and computational fluid dynamics (CFD). The thickness and surface coverage of PAO1 biofilms were observed to vary depending on the location in the flow cell and thus also the local wall shear stress. The biofilm structure in a 5:1 (width to height) aspect ratio rectangular flow cell was observed to consist mainly of a layer of bacterial cells with thicker biofilm formation observed in the flow cell corners. For square cross-section (1:1 aspect ratio) flow cells, generally thicker and more uniform surface coverage biofilms were observed. Mushroom shaped structures with hollow centers and wall breaks, indicative of ‘seeding’ dispersal structures, were found exclusively in the square cross-section tubes. Exposure of PAO1 biofilms grown in the flow cells to gentamicin revealed a difference in susceptibility. Biofilms grown in the rectangular flow cell overall exhibited a greater susceptibility to gentamicin compared to those grown in square flow cells. However, even within a given flow cell, differences in susceptibility were observed depending on location. This study demonstrates that the spanwise shear stress distribution within the flow cells has an important impact on the location of colonization and structure of the resultant biofilm. These differences in biofilm structure have a significant impact on the susceptibility of the biofilms grown within flow channels. The impact of flow modification due to flow cell geometry should be considered when designing flow cells for laboratory investigation of bacterial biofilms.     

临床分离铜绿假单胞菌对氟喹诺酮类抗菌药物的耐药性检测

目的了解临床分离铜绿假单胞菌对喹诺酮类等抗菌药物的耐药性。方法琼脂稀释法测定86株铜绿假单胞菌对5种氟喹诺酮类抗菌药物以及头孢吡肟、美罗培南的耐药性。结果铜绿假单胞菌对诺氟沙星、氧氟沙星、左氧氟沙星、环丙沙星、加替沙星的耐药率分别为50%、61.6%、51.2%、48.8%、51.2%;对头孢吡肟和美罗培南的耐药率分别为30.2%、23.2%。结论铜绿假单胞菌对氟喹诺酮类抗菌药物耐药显著,临床应加强检测和监测。     

Adherence of Pseudomonas aeruginosa to inanimate polymers including biomaterials

Cells of Pseudomonas aeruginosa were adhered to polymethyl methacrylate, polyvinyl acetate, polyvinyl chloride, polyhydroxyethyl methacrylate, mixed-acrylic, silicone, and natural latex materials. Planktonic bacteria and bacteria that adhered to the test materials were compared for their uptake of either L-[3,4,5-³H] leucine or [methyl-³H] thymidine during growth in a minimal medium. Leucine incorporation was reduced and thymidine uptake was negligible in adherent bacteria for up to 8 h following primary attachment by which time cells in the planktonic state showed active uptake of both substrates. These reduced uptake periods correlated with lag phases of growth of adherent cells as determined with a sonication-release plate count procedure and analyses of adenosine triphosphate (ATP). The extent of the lag phase of the adherent populations was dependent on initial densities of adhered cells and the nature of the substratum. Received 02 December 1998/ Accepted in revised form 25 April 1999     

铜绿假单胞菌生物膜悬液和藻酸盐对小鼠巨噬细胞吞噬功能的影响

目的探讨铜绿假单胞菌（PA）生物膜和藻酸盐成分对小鼠巨噬细胞吞噬功能的影响。方法用具有生物膜成分的PA悬液分别感染肺部巨噬细胞缺乏小鼠和正常小鼠,比较组织中的细菌数量。提取小鼠腹腔巨噬细胞,藻酸盐作用后加入PA悬液,测定巨噬细胞对细菌的吞噬率。巨噬细胞经不同浓度的藻酸盐作用后,中性红法检测巨噬细胞吞噬能力。结果巨噬细胞缺乏组和对照组肺部组织的细菌数量分别为（4.16±3.36）×10^5/ml和（5.15±1.92）×10^5/ml,t=0.7211,P=0.483。生物膜细菌组的巨噬细胞吞噬率与对照组的吞噬率分别为（13.82±4.71）%和（42.73±11.00）%,Q=12.3231,P〈0.01。表明生物膜细菌组比对照组更能抵抗巨噬细胞的吞噬。加藻酸盐组的巨噬细胞吞噬率与对照组的吞噬率分别为（22.91±6.20）%和（42.73±11.00）%,Q=8.4465,P〈0.01。表明加藻酸盐组比对照组更能抵抗巨噬细胞的吞噬。当藻酸盐浓度为0、25、50、75、100、125、150μg/ml时,以吸光度A（540nm）值表示其巨噬细胞吞噬中性红的能力分别为：0.271±0.044、0.456±0.062、0.445±0.061、0.551±0.065、0.210±0.053、0.186±0.026、0.195±0.025。当藻酸盐≤75μg/ml时巨噬细胞吞噬中性红的能力增强,与0μg/ml组相比P〈0.05;当藻酸盐〉75μg/ml时巨噬细胞吞噬中性红的能力降低,与0μg/ml组相比P〈0.05。结论巨噬细胞有阻止PA入侵的作用。PA生物膜可以抑制巨噬细胞的吞噬。PA生物膜藻酸盐成分在〈75μg/ml时促进巨噬细胞的吞噬,而在较大剂量时抑制巨噬细胞的吞噬功能。